Bioreactor

ABSTRACT

A bioreactor for microbial conversion of at least one conversion substrate, which has a treatment zone to accommodate when in use a solution of at least one conversion substrate, a culture holding zone to accommodate when in use a microbial culture capable of metabolizing at least one conversion substrate, a source of primary growth substrate for the microbial culture, a first permeable membrane forming an interface between the treatment zone and the culture holding zone, and a second permeable membrane forming an interface between the source of primary growth substrate and the culture holding zone, the first permeable membrane being of a material which will allow passage of the at least one conversion substrate from the treatment zone to the culture holding zone whilst being impermeable to the microbial culture, the second permeable membrane being of a material permeable to the primary growth substrate but substantially impermeable to water; and a process of operating said bioreactor.

FIELD OF THE INVENTION

The present invention relates to a bioreactor, and a process foroperation of a bioreactor.

BACKGROUND OF THE INVENTION

Biological processes, in particular microbial conversions, areincreasingly being employed to perform a variety of useful roles.Examples of applications in which microbial conversions have beensuccessfully utilised include microbial synthesis, the treatment ofindustrial effluent, the removal of contaminants from contaminated soilsand groundwaters, and the treatment of contaminated gas streams forexample biotreatment of air-stripped streams such as petroleum additivesand the like.

Over the past decade, environmental concerns and regulatory trends haveincreased the need for means to remove contaminants from contaminatedsoils and groundwaters. Such soils and groundwaters may becomecontaminated through natural sources for example the leeching of saltsand/or minerals from surrounding rocks into the soil or groundwater, butmore commonly they become contaminated through the activities of man,with contaminants such as metals, particularly heavy metals (e.g.mercury, chromium, lead); organic compounds (e.g. solvents, petroleum,petroleum related products, pesticides), inorganic compounds (e.g.nitrates); micro-organisms (e.g. pathogenic bacteria and viruses) andradio active compounds (e.g. uranium), having entered soils andgroundwaters through disposal or spillage. One such class ofcontaminants are branched alkyl ethers such as methyl tert-butyl ether(MTBE) which have been widely used as additives in gasoline blends andwhich have been found to accumulate in aquifer groundwaters socontaminating supplies of drinking water.

Accumulation of contaminants in soils and groundwaters may occur whennatural attenuation of said contaminants is limited by a lack ofsuitable microbes, oxygen, or nutrients (e.g. inorganic phosphate andnitrogen sources). One way to treat contaminated soils and groundwatersis to contact the soils or groundwaters with a microbial culture capableof converting the contaminant into non-harmful products in a bioreactor.Bioreactors have commonly been used in conjunction with ex-situpump-and-treat methods of bioremediation, wherein contaminatedgroundwater is pumped from an aquifer, treated with a microbial culturein a bioreactor, and reinjected into the aquifer or dischargedabove-ground. An alternative method of bioremediation is to employ anin-situ method, wherein an indigenous or non-indigenous microbialculture capable of metabolising a contaminant is injected into soils orgroundwaters contaminated with said contaminant in an environmentconducive to the growth and development of the culture. However, in-situmethods of bioremediation have proven difficult to control since theconditions in an aquifer are subject to change (e.g. pH, oxygenavailability, nutrient availability) making it difficult to create anenvironment conducive to the growth and development of the microbialculture. Further drawbacks of in-situ methods of bioremediation are thepotential for escape of non-indigenous microbial cultures from treatmentareas, and the blocking of the aquifer with solids.

To date, the practicality of using microbial cultures to remediatecontaminated soils and groundwaters has been limited in that many of themost persistent contaminants are metabolised very slowly as primarysubstrates and are only substantially metabolised at a satisfactory ratein co-metabolic microbial conversions. Microbial conversion of aparticular substrate by a microbial culture will usually involve bothcatabolic and anabolic activities, wherein the microbial cultureproduces multi catabolic enzymatic activities that degrade the compoundto intermediates which can either be used for biosynthetic purposes(anabolism) or can be mineralized to carbon dioxide as part of theenergy-generating processes of a cell. In such conventional microbialconversions, it is usually the degradation of the so-called primarygrowth substrate that provides energy and a carbon source for microbialgrowth. In contrast, a co-metabolic microbial conversion will involvethe catalytic activity of a few, often only a single, enzyme and theintermediates produced are not sufficiently available or suitable forthe key anabolic processes required to support microbial growth. Becauseof this separation between catabolic activity and anabolic growth, aprimary growth substrate must be supplied to support growth of microbialcultures for co-metabolic conversions, the primary growth substratebeing a carbon source which the culture can use to support its growth.To date, co-metabolic microbial conversions have been consideredunsuitable for use in in-situ methods of bioremediation as it is oftenimpractical and/or undesirable to add a primary growth substrate intoaquifer groundwater.

A further limitation on the use of co-metabolic microbial conversions isthat the primary growth substrate can be toxic to the microbial culturewhen provided at too high a concentration. For example, when the primarygrowth substrate is a hydrocarbon and the microbial culture exists in anaqueous medium, the addition of the hydrocarbon growth substrate at aconcentration greater than the solubility of the hydrocarbon in watercan kill culture cells. This is especially problematic as a high supplyrate and/or concentration of growth substrate is often required in orderto maintain a biomass of microbial culture sufficient to metabolise thequantity of substrate, e.g. contaminant, to be treated, and that thebioreactors presently available are inadequate for the maintenance of amicrobial culture in such circumstances.

U.S. Pat. No. 5,227,136 discloses a bioreactor vessel comprising a tankadapted to receive and contain a slurry, a mechanical mixing meansfitted in the tank, an air supply means which involves the introductionof minute air bubbles near the bottom region of the tank by a pluralityof elastic membrane diffusers (col. 3, line 20 to 32) and a means ofre-circulating exhaust gas stream back into the reactor contained slurryby means of the diffusers (col. 4, line 6 to 11). In use, slurrycontaining minerals, soils and/or sludges which have been contaminatedby toxic organic substances are delivered to the tank where they aredirectly contacted with and degraded by a biomass. Maintaining a highbiomass concentration in the reactor is said to be a task requiring theuse of equipment ancillary to the bioreactor (col. 4, lines 1 to 5) andin a preferred embodiment of the invention a biomass-carrying medium isadded to the slurry contained in the tank to assist in maintaining amaximum biomass concentration (col. 10 lines 10 to 16). Whilst it ismentioned at column 9, lines 54 to 57 that another gas which may act asa co-metabolite in a biodegradation process may be added with there-circulating gas stream, from the teaching of U.S. Pat. No. 5,227,136the skilled person would conclude that in operation it is necessary toprovide further microbial culture to the bioreactor vessel together witheach batch of slurry to be remediated.

WO 96/34087 discloses a bioreactor comprising a component definingchamber in which are disposed fluid treatment cells, a liquid-permeablemembrane which separates the chamber from a first channel in which fluidto be treated flows, and a gas-permeable membrane separating the chamberfrom a second channel in which an oxygenous gas flows. The chamberpreferably comprises two cell layers separated by a permeable layer. Thebioreactor is for the study of cell cultures, in particular tissue cellcultures, and is particularly developed for use in specialist medicalapplications such as an artificial liver or a device for facilitatingthe functioning of a failing liver. Whilst the bioreactor of WO 96/34087comprises two separate membranes through which oxygen and fluid to betreated may be simultaneously supplied to the cells, there is no mentionof a separate means to supply the treatment cells with a primary growthsubstrate (i.e. a source of carbon and energy) other than the fluid tobe treated. Therefore, from the teaching of WO 96/34087 a skilled personwould be led to conclude that the bioreactor disclosed therein issuitable only for conventional or non co-metabolic microbialconversions, in particular conversions using tissue cultures.

It is apparent therefore that there is a need for a bioreactor which maybe used with both conventional microbial conversions and co-metabolicmicrobial conversions and which may be adapted for use in in-situmethods of bioremediation. cl SUMMARY OF THE INVENTION

The present invention provides a bioreactor for microbial conversion ofat least one conversion substrate which comprises a treatment zone toaccommodate when in use a solution of said at least one conversionsubstrate, a culture holding zone to accommodate when in use a microbialculture capable of metabolising said at least one conversion substrate,a source of primary growth substrate for the microbial culture, a firstpermeable membrane forming an interface between the treatment zone andthe culture holding zone, and a second permeable membrane forming aninterface between the source of primary growth substrate and the cultureholding zone, the first permeable membrane being of a material whichwill allow passage of the at least one conversion substrate from thetreatment zone to the culture holding zone whilst being impermeable tothe microbial culture, the second permeable membrane being of a materialpermeable to the primary growth substrate but substantially impermeableto water.

BRIEF DESCRIPTION OF DRAWINGS

The invention will now be further illustrated by way of example withreference to the accompanying schematic drawings.

FIG. 1 is a schematic depiction of a first embodiment of the presentinvention in the form of an apparatus for batch microbial conversionwherein the first and second permeable membranes are in the form of twoannular tubes, one located inside the other,

FIG. 2 is a sectional perspective view of the first and second permeablemembranes of the first embodiment,

FIG. 3 is a cross-section of FIG. 2 along the line X to X,

FIG. 4 is a vertical cross-section of a second embodiment of the presentinvention, wherein the bioreactor is a well-type dual membrane reactoradapted for use in continuous bioremediation of aquifer groundwater inan aquifer,

FIG. 5 is a cross-section of the second embodiment of FIG. 4 along theline X′ to X′,

FIG. 6 is a perspective view of a third embodiment of the presentinvention, wherein the bioreactor is a parallel-sheet membrane reactoradapted for use in continuous bioremediation of aquifer groundwater inan aquifer,

FIG. 7 is a vertical cross-section of the third embodiment of FIG. 6,

FIG. 8 is a further vertical cross-section of the third embodiment whichis normal to the cross-section of FIG. 7 and which dissects a treatmentzone along a line “X,” to X″.

FIG. 9 is a plan view of an area of ground containing a gated barriertreatment system for incorporating the second embodiment of FIGS. 4 and5, and the third embodiment of FIGS. 6, 7 and 8.

DETAILED DESCRIPTION OF THE INVENTION

A bioreactor according to the present invention may be used for themicrobial conversion of at least one conversion substrate in solutionfor a variety of different applications. For example, a bioreactoraccording to the present invention may conveniently be used for theco-metabolic microbial conversion of contaminants in contaminated soilsand groundwaters, a preferred bioreactor according to the presentinvention being a bioreactor for the treatment of water contaminatedwith at least one contaminant. In such an instance, the or eachcontaminant is a conversion substrate, and the term treatment ofcontaminated water does not include the treatment of water-based bodyfluids in medical treaments.

In another example, a bioreactor according to the present invention maybe used for the treatment of at least one contaminant in a gas phasesolution i.e. for the removal of contaminants from gas streams, e.g.volatile organic compounds from air or hydrogen sulphide from naturalgas.

Co-metabolic microbial conversions are useful in the treatment ofcontaminants which are not readily metabolised by indigenous microbialcultures and therefore accumulate in groundwaters. One such class ofcontaminants are branched alkyl ethers, for example methyl tert-butylether (MTBE), tert-amyl methyl ether (TAME), ethyl tert-butyl ether(ETBE), and di-isopropyl ether (DIPE), which have been used in gasolineblends as lead replacement additives. These contaminants, which mayenter the groundwater through accidental spills and the re-deposition ofchemicals emitted to the atmosphere from partially combusted automobileexhaust, accumulate in the groundwater as they are both soluble in waterand slow to degrade. In particular, there are concerns over thecontamination of drinking wells with MTBE and its primary metabolitetert-butyl alcohol (TBA). In particular, MTBE has an unpleasant taste,which is recognisable in concentrations in drinking water at a parts perbillion level.

Accordingly, the present invention further provides a bioreactor for thetreatment of water contaminated with at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols whichcomprises a treatment zone to accommodate when in use contaminatedwater, a culture holding zone to accommodate when in use a microbialculture capable of metabolising the at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols, asource of hydrocarbon growth substrate for the microbial culture, afirst permeable membrane forming an interface between the treatment zoneand the culture holding zone, and a second permeable membrane forming aninterface between the source of hydrocarbon growth substrate and theculture holding zone, the first permeable membrane being of a materialwhich will allow passage of the at least one alkoxy compound selectedfrom branched alkyl ethers and branched alkyl alcohols but impermeableto the microbial culture, the second permeable membrane being permeableto the hydrocarbon growth substrate but impermeable to water.

The first and second permeable membranes of the present invention may beprovided in any convenient form and in any convenient configuration. Forexample, the first and second permeable membranes may each independentlytake the form of at least a part of one or more walls of a vessel, alength of tubing, or one or more sheets of permeable membrane. The firstand second permeable membranes may be of a similar form, (e.g. twolengths of tubing, one being located inside the other) arranged about acentral length-wise axis in an optionally co-axial or co-linearconfiguration, or alternatively the first and second permeable membranesmay be of a dissimilar form, e.g. a first permeable membrane in the formof at least a part of one or more walls of a vessel and the secondpermeable membrane in the form of tubing, the tubing of the secondpermeable membrane being positioned within the vessel of the firstpermeable membrane. Further, the first and/or second permeable membranemay be in the form of a plurality of sheets arranged within a supportingframework. In a preferred embodiment of the present invention the firstand second permeable membranes are each independently in the form of alength of tubing, the tubing of the second permeable membrane beinglocated inside the tubing of the first permeable membrane.

Preferably the first and second permeable membranes are provided in aconfiguration wherein the microbial culture is substantially confinedwithin the culture holding zone such that microbial culture cannotescape from the culture holding zone into the treatment zone.Accordingly, a preferred bioreactor according to the present inventionis a bioreactor wherein the culture holding zone is defined by theboundaries of a chamber, the treatment zone is external of the chamberand the second permeable membrane forms at least a part of a hollowmember situated within the chamber. The boundaries of said chamber mayconveniently be defined solely by first permeable membrane however theymay be partly defined by first permeable membrane and partly defined byimpermeable material.

A bioreactor according to the present invention may conveniently be usedfor both batch and continuous microbial conversion of an at least oneconversion substrate in solution. Further, the bioreactor mayconveniently be used for the treatment of contaminated groundwater inconjunction with pump-and-treat methods of bioremediation and, bysuitable location of the bioreactor, in in-situ methods ofbioremediation. When the bioreactor is for batch treatment of an atleast one conversion substrate in solution, e.g. for the treatment of abatch of groundwater contaminated with at least one contaminant, it isconvenient for the bioreactor to further comprise an impermeabletreatment vessel which defines the boundaries of the treatment zone.

The first permeable membrane may be of any material that will allowpassage of the at least one conversion substrate from the treatment zoneto the culture holding zone whilst being impermeable to the microbialculture.

Examples of materials which may, depending on the nature of theconversion substrate, as will be readily understood by those skilled inthe art, be utilised as first permeable membrane of the presentinvention include porous or sintered materials (e.g. glass, ceramics,and metals), porous plastics, cellulose acetate, low densitypolyethylene, polyisoprene (natural rubber), polyvinylidene chloride,polyamide, polyethylene terephthalate, polysulfones, polyvinylidenefluoride, polydialkylsiloxane (silicone rubber) and fluorinatedethylene-propylene copolymer;

The permeability of a membrane is dependant upon both the chemicalnature of a membrane material (e.g. its diffusion co-efficient andchemical selectivity (e.g. hydrophobicity, hydrophilicity, etc.)) andphysical factors relating to the construction of the membrane e.g.thickness, pore size and shape.

Types of membranes which may conveniently be employed in the presentinvention include reverse osmosis membranes, e.g. composite membranescomprising a layer of a first material on top of a second material (e.g.polyamide on top of a polysulfone filler), typically having a pore sizeof from 4×10⁻⁴ μm to 6×10⁻² μm; ultra and microfiltration membranes,e.g. membranes of cellulose acetate and plastic materials e.g.polysulfones, polyvinylidene fluoride and fluorinated ethylene-propylenecopolymer, typically having a pore size of from 2×10⁻³ μm to 10 μm andreticulated materials and screens having a pore size of from 10 μm to3×10³ μm.

The first permeable membrane is preferably a membrane having a highpermeability with regard to the at least one conversion substrate as thefaster the at least one conversion substrate may pass through the firstpermeable membrane the greater the versatility of the bioreactor.Preferably, the specific surface area of the first permeable membrane ofthe bioreactor is no less than 10 m² per m³ of reactor, and ispreferably in the range of from 10 to 1000 m²/m³, more preferably in therange of from 25 to 500 m²/m³ and most preferably in the range of from50 to 300 m²/m³. The higher the specific surface area of first permeablemembrane the greater the versatility of the bioreactor.

The first permeable membrane may be permeable to the solution of atleast one conversion substrate or it may be selective in that it ispermeable to a solute only, i.e. permeable to the at least oneconversion substrate but substantially impermeable to a solvent of thesolution. For example, when the bioreactor is a bioreactor for thetreatment of water contaminated with at least one contaminant the firstpermeable membrane may be permeable to the at least one contaminant butsubstantially impermeable to water.

Examples of membranes permeable to water include cellulose acetate,reticulated foams and porous or sintered materials (e.g. membranes madefrom glass, ceramics, and metals) and porous plastics membranes (e.g.membranes made from plastics having a pore size and chemical selectivitysuch that they will allow passage of water).

A preferred bioreactor according to the present invention is abioreactor wherein the first permeable membrane is substantiallyimpermeable to water. Examples of membranes substantially impermeable towater include membranes made from low density polyethylene, polyisoprene(natural rubber), polyvinylidene chloride, polyamide, polyethyleneterephthalate, polydialkylsiloxane (silicone rubber) and fluorinatedethylene-propylene copolymer. As will be understood by those skilled inthe art, membranes that are substantially impermeable to water will beof low porosity and high hydrophobicity. Examples of preferred firstpermeable membranes which are substantially impermeable to water areavailable from Watson Marlow Ltd., under the trade mark “Marprene”(silicone rubber); from Du Pont Ltd., under the trade mark “Viton”(fluoroelastomer) and from Norton Performance Plastics Ltd., under thetrade mark “Tygon” (silicone rubber).

The first permeable membrane of the present invention may or may not bepermeable to the primary growth substrate, however in circumstanceswhere the release of primary growth substrate from the bioreactor isundesirable it is preferred that the first permeable membrane besubstantially impermeable to the primary growth substrate. Further,where the metabolism of the at least one conversion substrate provides aconversion product unsuitable for release from the bioreactor (e.g.where methyl-tert butyl ether (MTBE) is converted to t-butyl alcohols(TBA)), it is preferred that the first permeable membrane is impermeableto that conversion product, whilst when a conversion product is suitablefor release from the bioreactor (e.g. carbon dioxide) it is preferredthat the first permeable membrane is of a material permeable to thatconversion product.

A preferred bioreactor according to the present invention is abioreactor wherein the conversion substrate is at least one organiccompound, for example solvents and petroleum related products such asgasolines, BTEX (i.e. benzene, toluene, ethylbenzene and xyleneisomers), ethanol and ether oxygenates and their derivatives. Morepreferably the conversion substrate is at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols e.g.methyl tert-butyl ether (MTBE), tert-amyl methyl ether (TAME), ethyltert-butyl ether (ETBE), diisopropyl ether (DIPE) and tert-butyl alcohol(TBA), and most preferably the conversion substrate comprises methyltert-butyl ether (MTBE).

Examples of materials which may very conveniently be used as firstpermeable membrane when the at least one conversion substrate is an atleast one alkoxy compound selected from branched alkyl ethers andbranched alkyl alcohols, e.g. MTBE, include polydialkylsiloxane(silicone rubber), low density polyethylene, polyisoprene (naturalrubber), and porous plastics materials.

The second permeable membrane may be of any material permeable to theprimary growth substrate but substantially impermeable to water and ispreferably impermeable to the microbial culture. Preferably the secondpermeable membrane is of a material having a high permeability withregard to the primary growth substrate as the faster the primary growthsubstrate may permeate through the second permeable membrane the greaterthe amount of primary growth substrate that can be supplied to themicrobial culture through a given surface area of second permeablemembrane and the greater the versatility of the bioreactor.

The primary growth substrate may be any carbon and energy containingsubstrate on which the culture may grow and may conveniently be anorganic compound. Examples of organic primary growth substrates whichmay be utilised to support the growth of a microbial culture include forexample alcohols e.g. ethanol and methanol; ethers e.g. diethyl ether;esters; acids e.g. ethanoic acid and propionic acid; sugars e.g.glucose; and hydrocarbons including aromatic hydrocarbons e.g. tolueneand benzene, alkenes, e.g. ethene; and alkanes e.g. methane, ethane,propane, butane, pentane etc. and cycloalkanes e.g. cyclohexane, andmixtures thereof.

When the bioreactor of the present invention is a bioreactor for thetreatment of water contaminated with at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols, theprimary growth substrate is preferably at least one hydrocarbon growthsubstrate of up to 12 carbon atoms e.g. decane, dodecane; morepreferably at least one hydrocarbon growth substrate of up to 9 carbonatoms e.g. benzene, toluene, ethylbenzene, xylene. Even more preferablythe primary growth substrate is at least onealkane of up to 6 carbonatoms, e.g. methane, ethane, propane, butane, pentane, hexane andcyclohexane; and is preferably an alkane of from 2 to 6 carbon atoms,more preferably of from 3 to 6 carbon atoms. An example of a hydrocarbongrowth substrate which may very conveniently be employed is cyclohexane.

Examples of materials which may, depending on the nature of the primarygrowth substrate, as will be readily understood by those skilled in theart, be utilised as second permeable membrane include low densitypolyethylene, polyisoprene (natural rubber), polyvinylidene chloride,polyamide, polyethylene terephthalate, polydialkylsiloxane (siliconerubber), polysulfones and polyvinylidene fluoride and fluorinatedethylene-propylene copolymer. Examples of preferred second permeablemembranes are available from Watson-Marlow Ltd., under the trade mark“Marprene” (silicone rubber); from Du Pont Ltd. under the trade mark“Viton” (fluoroelastomer) and from Norton Performance Plastics Ltd.under the trade mark “Tygon” (silicone rubber).

A preferred bioreactor according to the present invention is abioreactor wherein the second permeable membrane is of a materialpermeable to organic compounds, more preferably of a material permeableto hydrocarbons, even more preferably of a material permeable toalkanes, and most preferably of a material permeable to cycloalkanes,e.g. cyclohexane. Examples of materials which may conveniently be usedas second permeable membrane when the primary growth substrate is analkane include polydialkyl siloxane (silicone rubber) and low densitypolyethylene. Conveniently, the second permeable membrane is of amaterial permeable to oxygen and nutrient sources, e.g. ammonia, so thatadditional oxygen and nutrient sources may be provided to the microbialculture together with the primary growth substrate. Advantageously, thesecond permeable membrane is of a material permeable to carbon dioxideso that when microbial conversion provides carbon dioxide as aconversion product the carbon dioxide may exit the culture holding zonethrough the second permeable membrane and be removed from the bioreactortogether with any excess primary growth substrate. A build up of carbondioxide in the culture holding zone may be harmful to the microbialculture.

The primary growth substrate may be supplied from the source of primarygrowth substrate to the second permeable membrane as a liquid or a gas.The primary growth substrate may be supplied continuously or it maysupplied intermittently. Preferably the primary growth substrate issupplied continuously.

Conveniently, the primary growth substrate may be supplied as a gas,which gas may comprise neat primary growth substrate or a mixture ofprimary growth substrate and an oxygen-containing gas e.g. air oroxygen. When the primary growth substrate is a gas, the second permeablemembrane may advantageously be of a material having a high diffusionco-efficient with respect to the primary growth substrate. When theprimary growth substrate is a liquid, the second permeable membrane mayadvantageously be of a material which does not expand or swell whensaturated with primary growth substrate.

The supply of a gaseous primary growth substrate to the microbialculture through a permeable membrane is advantageous as it allowsamounts of growth substrate to be delivered to the microbial culturewhich if delivered directly may have a toxic effect upon the microbialculture. The ability to supply a larger amount of primary growthsubstrate allows a higher biomass of microbial culture to be grown upquickly and to be maintained.

The source of primary growth substrate may comprise a store of primarygrowth substrate in direct communication with the second permeablemembrane or it may comprise a store of primary growth substrate and adelivery means for the supply of primary growth substrate from the storeto the second permeable membrane. The delivery means may comprise, forexample, manifold means, channels, conduits, and tubes, and a pumpingmeans. The bioreactor may further comprise a control means to regulatethe amount of primary growth substrate delivered to the first permeablemembrane and an exhaust means for the removal of excess primary growthsubstrate from the bioreactor.

In addition to a growth substrate, certain microbial cultures requirenutrients such as inorganic phosphates, nitrogen sources (e.g. NH₄ ⁺,NO₃ ⁻) and micro-nutrients (e.g. sources of magnesium, calcium,potassium, iron, manganese, molybdenum, boron, copper and zinc) in orderto grow. Whilst adequate amounts of such nutrients may be available inthe solution of at least one conversion substrate to be treated e.g. incontaminated groundwater, the bioreactor of the present invention mayadvantageously comprise a means of supplying nutrients and/or additionalmicrobial culture to the culture holding zone, circulating nutrientand/or additional microbial culture through the culture holding zone,and removing waste nutrient and/or microbial culture from the cultureholding zone, which means may, for example, comprise manifold means,channels, conduits, tubes and the like, and a pumping means for thedistribution or circulation of nutrient and/or microbial culture throughthe culture holding zone. The circulation of a nutrient solution throughthe culture holding zone is advantageous as it allows a means by whichthe temperature, pH and dissolved oxygen content of the media within theculture holding zone may be conveniently monitored. Accordingly, apreferred bioreactor according to the present invention is a bioreactorfurther comprising pH and/or temperature, and/or dissolved oxygencontent controls associated with the means of supplying and circulatingnutrient and/or microbial culture. It will be understood that when thenutrient is supplied as an aqueous solution it is preferable that thefirst permeable membrane is substantially impermeable to water.

The microbial culture in the culture holding zone may be any microbialculture capable of performing the desired microbial conversion. Themicrobial culture may be a substantially pure microbial culturecomprising a single species of microbe or it may be a mixed microbialculture comprising at least two species of microbes. The microbialculture may be selected from bacteria, fungi, algae, protozoa andmixtures thereof and may be an aerobic or anaerobic microbial culture.Preferably the microbial culture is an aerobic microbial culture.

Microbial conversions with mixed bacterial cultures are commonlyco-metabolic in that the mixed culture will comprise microbes capable ofmetabolising the primary growth substrate to metabolites and/or enzymesand further microbes which are capable of performing the desiredmicrobial conversion and which require said metabolites and/or enzymesfor growth. When the bioreactor of the present invention is used forin-situ bioremediation of contaminated groundwaters, i.e. wherein thebioreactor is located in an aquifer containing the contaminated water,the microbial culture employed in a bioreactor of the present inventionmay be an indigenous culture, collected from the contaminated site andoptionally enriched, or a non-indigenous culture. Preferably themicrobial culture is a non-indigenous culture. Conveniently, themicrobial culture may be a hydrocarbon-utilising culture, preferably analkane-utilising culture, more preferably a cycloalkane-utilisingculture, e.g. a cyclohexane-utilising culture. When the bioreactor ofthe present invention is a bioreactor for treatment of watercontaminated with at least one contaminant the microbial culture ispreferably a culture capable of metabolising the contaminant to carbondioxide and water.

The microbial culture may grow as a biofilm on the membrane surface ofthe first and/or the second permeable membrane or may be at leastpartially suspended within a medium within the culture holding zone. Thebioreactor may further provide means for fixing or otherwise supportingthe microbial culture by providing within the culture holding zone asuitable support, for example in the form of a scaffold, porous sponge(e.g. reticulated foam), or a matrix or packing (e.g. glass or plasticbeads) to which the microbial population can adhere, the supportpreferably being of a material conducive to the formation of biofilms.

A biomass of microbial culture required to perform the microbialconversion may be established in the culture holding zone by adding asample of microbial culture to the culture holding zone and growing thesample until a required biomass of microbial culture is established oralternatively the required biomass of microbial culture may be grownseparately and introduced into the culture holding zone in-toto.

It is preferred that once a required biomass of microbial culture hasbeen established in the culture holding zone that no further culture beadded to the bioreactor and the biomass of culture be sustained by thesupply of primary growth substrate alone. However, the reactor ofpresent invention may advantageously further incorporate means for theaddition of further microbial culture to the culture holding zone, andthe removal of dead cells and waste media from the culture holding zone.

Details of known microbial cultures capable of co-metabolic conversionof organic contaminants are given in Table 1. Details of microbialcultures capable of co-metabolic conversion at least one branched alkylether are given in Table 2.

TABLE 1 Primary Growth Conversion Substrate Substrate ReferenceChlorobenzene Trichloroethylene C.M. Kao et al. Jn. Hazardous Materials,1999, pp. 67-69 Ethene cis-1,2-dichloroethene P. Koziollek et al. andvinyl chloride Arch Microbiol, 172 1999, pp. 240-246 Ethenetrichloroethylene C.E. Aziz et al. Biotechnology and Bioengineering, 65,1999, pp. 100-107 Butane trichloroethylene F.A. Perriello et al.,Journal of Soil Contamination, 8 (1), 1999, pp. 117-129 Toluenetrichloroethylene C.C. Cox et al. Wat. Sci. Tech., 37, 1998, pp. 97-104Ethanol 2,4-dinitrotoluene J.Y. Cheng et al. Wat. Res., 30, 1996, pp.307-314 Methane trichloroethylene P.L. McCarty, et al. Biotechnology andBioengineering, 55, 1997, pp. 650-659 Butane chloroform N. Hamamura etal. Applied and Environmental Microbiology, 65, 1999, pp. 4586-4593

TABLE 2 Primary Growth Conversion Substrate Substrate Reference Pentanemethyl tert-butyl P.M. Garnier et.al. ether Appl. Microbiol Biotechnol.,51, 1999, pp. 498-503 Propane methyl tert-butyl R.J. Steffan et al.ether, Applied and ethyl tert-butyl Environmental ether, Microbiology,63, 1997, tert-amyl methyl pp. 4216-4222 ether Diethyl Ether methyltert-butyl L.K. Hardison et.al. ether Applied and EnvironmentalMicrobiology, 63, 1997, pp. 3059-3067 Cyclohexane methyl tert-butyl D.Corcho-Sanchez et ether* al. Co-metabolic degradation of MTBE by acyclohexane-oxidising bacteria, Proceedings of Battelle conference:Remediation of chlorinated and recalcitrant Compounds: Monterey,California, May 22-25, 2000. *N.B. in this reference metabolism ceasesat conversion of MTBE to TBA.

In one preferred embodiment of the present invention the bioreactor maybe a bioreactor adapted for the batch treatment of at least oneconversion substrate.

In accordance with this preferred embodiment the bioreactor preferablycomprises a first permeable membrane in the form of tubing and a secondpermeable membrane in the form of tubing, the tubing of the secondpermeable membrane being located inside the tubing of the firstpermeable membrane in a configuration which may or may not be co-axialto the first permeable membrane, and a treatment vessel, in which vesselthe tubing of the first and second permeable membranes are located. Thetubing of the first and second permeable membranes may be any type oftubing which may be practically employed however it may conveniently beannular tubing. The tubing of the first and second permeable membranesmay be arranged in the treatment vessel randomly, as helices or in anyother configuration which allows for a required surface area of first orsecond permeable membrane to be incorporated into the bioreactor.

The tubing of the second permeable membrane of this preferred embodimentis further connected to a store of primary growth substrate and anexhaust means whilst the tubing of the first permeable membrane mayconveniently be further connected to a store of nutrient and/ormicrobial culture and an effluent means. Advantageously, the store ofnutrient and/or microbial culture may be associated with control meanswith which to regulate the conditions in the culture holding zone e.g. apH control, a temperature control or a dissolved oxygen control.

The first permeable membrane of this preferred embodiment is preferablyof a material permeable to organic contaminants (e.g. branched alkylethers and/or branched alkyl alcohols) but substantially impermeable towater e.g. polydialkylsiloxane (silicone rubber). The second permeablemembrane may conveniently be made of the same material as the firstpermeable membrane e.g. polydialkylsiloxane (silicone rubber).

For greater efficiency dimensions of the tubing of the first and secondpermeable membranes of this preferred embodiment should be optimised byexperimentation in a manner which will be understood by those skilled inthe art, to obtain maximum performance for the application in which itis to be used. However, the tubing of the first permeable membrane mayconveniently be annular tubing having an outside diameter of from 3 mmto 20 mm, preferably of from 4 mm to 15 mm and most preferably of from 5mm to 10 mm, and a wall thickness of of from 0.01 mm to 2 mm, preferablyof from 0.1 mm to 1.5 mm and most preferably of from 0.2 mm to 1.0 mm.The tubing of the second permeable membrane may conveniently be annulartubing having an outside diameter of from 1 mm to 19 mm, preferably offrom 2 mm to 14 mm and most preferably of from 3 mm to 9 mm; and a wallthickness of from 0.01 mm to 5 mm, preferably of from 0.1 mm to 4 mm,and most preferably of from 0.2 mm to 3 mm.

An advantageous feature of this preferred embodiment is that the firstand second membranes may conveniently be pre-prepared as a continuouslength of dual membrane tubing and that a required length of tubing maybe conveniently cut from said continuous length of tubing to suit theapplication for which the bioreactor is to be used. Advantageously, saidcontinuous lengths of tubing may be provided with at least one separatormeans located between the first and second permeable membranes so as tomaintain a gap between the membranes. The or each separator may bepositioned at intervals or may run lengthwise throughout the continuouslength of tubing.

Whilst the bioreactor of this preferred embodiment is described above asbeing adapted for batch treatment of at least one conversion substrateit will be understood by those skilled in the art that this embodimentmay conveniently be adapted for use in in-situ treatment of an at leastone conversion substrate for example by location of at least one lengthof dual membrane tubing in an aquifer containing contaminated water. Forexample, a bioreactor according to the present invention mayconveniently comprise a top-connection unit, a bottom-connection unitand a plurality of lengths of dual membrane tubing which are positionedbetween said top-connection unit and bottom-connection unit, thetop-connection unit being fitted with connections to a source of primarygrowth substrate and a source of nutrient and/or microbial culture andwith connections to escape and exhaust means through which excessprimary growth substrate and nutrient and/or microbial culturerespectively can exit the bioreactor, wherein in use this bioreactor maybe positioned in an aquifer bore-hole and primary growth substrate andoptionally nutrient and/or microbial culture supplied to thetop-connection unit, distributed via the top-connection unit to aproportion of the lengths of dual membrane tubing, passed down therespective primary growth substrate supply paths and culture holdingzones of the proportion of lengths of dual membrane tubing to thebottom-connection unit, distributed via the bottom-connection unit tothe remaining lengths of tubing, and passed up the remaining lengths oftubing to the top-connection unit and out of the bioreactor via theescape and exhaust means respectively.

The bioreactor of the present invention may conveniently be located inan aquifer containing contaminated water, for employment in an in-situmethod of bioremediation. When used for in-situ bioremediation, thebioreactor is preferably located within a receptacle through whichgroundwater is free to flow and which may be conveniently positionedwithin, and removed from, an aquifer. Preferably, the aquifer is fittedwith means to direct the flow of groundwater within the aquifer towardsthe receptacle, the treatment zone being an area within the receptacle.

U.S. Pat. No. 5,487,622, herein incorporated by reference, describes agated-barrier system for treating polluted groundwater in which abioreactor according to the present invention may be conveniently used(see column 5, lines 27-42), wherein contaminated groundwater is treatedin-situ by funneling groundwater through a gate or gates in a watertightin-ground wall of metal sheets which are pile driven into the ground,which gate or gates are so constructed so as to allow the groundwater topass there-through and hence through the water-tight wall and which gateor gates contain a receptacle formed as a container for containing abody of treatment material (i.e. a bioreactor according to the presentinvention), and yet allowing the contaminated water to pass into thereceptacle for which purpose the receptacle is provided with slots.

Accordingly in a further preferred embodiment of the present inventionthe bioreactor may be a well-type dual membrane reactor adapted for usein continuous bioremediation of aquifer groundwater in an aquifer.

In accordance with this further preferred embodiment the bioreactorpreferably comprises a vessel formed from a first permeable membrane anda second permeable membrane in the form of tubing positioned within thevessel. The vessel formed from the first permeable membrane may be ofany practical shape but may conveniently be a cylindrical vessel, inwhich case either the whole vessel may be made from the first permeablemembrane, or the cylindrical walls alone or the circular base alone maybe made of the first permeable membrane, the base or the walls partiallybeing made of an impermeable material. Conveniently, the tubing of thesecond permeable membrane is annular tubing. The tubing of the secondpermeable membrane may be arranged randomly, as one or more helices, asa spiral-wind, or in any other configuration which allows for a requiredsurface area of second permeable membrane to be incorporated into thereactor. The tubing of the second permeable membrane is furtherconnected to a store of primary growth substrate and an exhaust means.

The vessel of the first permeable membrane of this further preferredembodiment may be made of any material that will allow passage of an atleast one contaminant, and is preferably located in a receptacle intowhich the groundwater is channelled and through which it may flow. Whenthe first permeable membrane is of a material that is permeable to theat least one contaminant but substantially impermeable to water thevessel is conveniently arranged in the receptacle such that groundwatermay freely flow around the vessel. Alternatively, when the firstpermeable membrane is of a material permeable to water the receptaclemay be conveniently be further provided with impermeable stoppers placedwithin the receptacle to channel the groundwater through the firstpermeable membrane. The second permeable membrane may be of any materialpermeable to the primary growth substrate.

Advantageously this further preferred embodiment may further comprise agas-diffuser located within the culture holding zone through whichoxygen-containing gas may be directly supplied to the culture holdingzone. The oxygen-containing gas delivered through the gas diffuser mayadvantageously be used to agitate media within the culture holding zoneand to assist in pH and temperature control. Alternatively a mixer maybe incorporated to agitate the media.

For greatest efficiency, dimensions of this further preferred embodimentshould be optimised, by experimentation in a manner which will beunderstood by those skilled in the art, to obtain maximum performancefor the application and aquifer in which it is to be employed. Howeverwhen the vessel of first permeable membrane is a cylindrical vessel itmay conveniently have an outside diameter of from 0.01 m to 20 m,preferably of from 0.05 m to 5 m, most preferably of from 0.2 m to 2 m;and a wall thickness of from 0.10 mm to 100 mm, preferably of from 0.20mm to 50 mm, most preferably of from 0.50 mm to 20 mm; and have avertical height approximately 0.5 m greater than the distance from thebase of the aquifer to the highest level of the aquifer water table. Thetubing of the second permeable membrane may conveniently be annulartubing having an outside diameter of from 1 mm to 19 mm, preferably offrom 2 mm to 14 mm and most preferably of from 3 mm to 9 mm; and a wallthickness of from 0.01 mm to 5 mm, preferably of from 0.1 mm to 4 mm andmost preferably of from 0.2 mm to 3 mm.

Whilst the bioreactor of this further preferred embodiment is describedherein as being adapted for use in an in-situ method of bioremediation,it will be understood by those skilled in the art that this embodimentmay conveniently be adapted for use in ex-situ systems for treatment ofan at least one conversion substrate e.g. by location of the vessel andtubing positioned therein in a treatment vessel containing a solution ofat least one conversion substrate or through which a solution of atleast one conversion substrate may be passed.

When a bioreactor according to the present invention is to be located inan aquifer containing contaminated groundwater for employment in anin-situ method of bioremediation, it is advantageous for the surfacearea of first permeable membrane in contact with aquifer groundwater tobe as large as possible.

Accordingly in another preferred embodiment of the present invention thebioreactor may be a parallel sheet membrane reactor adapted for use incontinuous bioremediation of aquifer groundwater in an aquifer.

In accordance with this preferred embodiment the bioreactor maypreferably comprise a plurality of sheets of first permeable membrane, aplurality of second permeable membranes and a supporting frameworkpreferably comprising two side-walls, a plurality of rectangular framesand optionally a plurality of spacer means, the plurality of sheets offirst permeable membrane, plurality of rectangular frames and pluralityof spacer means being positioned parallel with respect to one anotherbetween the two side-walls, either substantially horizontally orsubstantially vertically, such that the bioreactor comprises a pluralityof treatment zones defined by boundaries of a channel formed between twoadjacent sheets of first permeable membrane, a rectangular frame and/ora spacer means; a plurality of culture holding zones defined byboundaries of a chamber formed between a sheet of first permeablemembrane, a second permeable membrane and a rectangular frame; and aplurality of primary growth substrate supply paths defined by boundariesof a chamber formed by a second permeable membrane and optionally arectangular frame. The second permeable membrane may be in the form oftubing, with a length of tubing passing through each culture holdingzone and each length of tubing defining the boundaries of a primarygrowth substrate supply path, or it may be in the form of sheets whereintwo sheets of second permeable membrane are positioned within eachculture holding zone which two sheets of membrane together with arectangular frame define the boundaries of a primary growth substratesupply path.

Advantageously each treatment zone of this preferred embodiment may befitted with a spacer means, each spacer means comprising at least oneaperture through which groundwater may enter a treatment zone, a flowpath through which groundwater may flow through a treatment zone, and atleast one exit aperture through which groundwater may exit a treatmentzone, e.g. downstream of the bioreactor. The flow path is preferablyrouted such that groundwater entering a treatment zone has maximumcontact with first permeable membrane before leaving the treatment zoneand/or maximum residence time within the treatment zone. Further, theculture holding zone and/or primary growth substrate supply paths may befitted with a separator means to maintain a sufficient gap between therelevant membranes.

The bioreactor of this preferred embodiment further comprises a meansfor supply of primary growth substrate from a store of primary growthsubstrate to each primary growth substrate supply path and willadvantageously comprise a means for supply of nutrient and/or microbialculture to the plurality of culture holding zones and an effluent means.The sheets of first permeable membrane are preferably permeable to theat least one contaminant but substantially impermeable to water. Thesecond permeable membrane may be of any material permeable to theprimary growth substrate and may conveniently be of the same material asthe first permeable membrane.

It is preferred that the sheets of first permeable membrane are spacedand are of a thickness so as to provide the bioreactor with the largestpossible area of contact between first permeable membrane andcontaminated water in the treatment zone whilst still allowing foradequate flow of the groundwater through the bioreactor and adequatesupply of growth substrate to, and accommodation of, the microbialculture. Accordingly, the thickness of each sheet of first or secondpermeable membrane will preferably be 3 mm or less, more preferably offrom 0.05 mm to 1 mm, most preferably of from 0.1 mm to 0.50 mm. Thewidth of each treatment zone, which may conveniently be maintained by aspacer means, will preferably be from 1 to 10 mm, more preferably from 2mm to 8 mm and most preferably from 2 mm to 5 mm. When the secondpermeable membrane is in the form of sheets of permeable membrane thewidth of each culture holding zone, is preferably from 1 to 6 mm, morepreferably of from 2 to 5 mm and and the width of each primary growthsubstrate supply path, is preferably from 0.2 mm to 2 mm, morepreferably 0.5 mm to 1 mm. When the second permeable membrane is in theform of a length of annular tubing positioned within each cultureholding zone the tubing is preferably annular tubing having an outsidediameter of from 1 mm to 6 mm, preferably of from 2 mm to 5 mm and mostpreferably of from 3 mm to 4 mm; and a wall thickness of from 0.01 mm to2.5 mm, preferably of from 0.1 mm to 2 mm and most preferably of from0.2 mm to 1.5 mm. When the supporting framework comprises a plurality ofrectangular frames, the width of a primary growth substrate path and aculture holding zone may conveniently correspond to the width of arectangular frame partially defining its boundaries, which frames mayconveniently be fitted with seals so as to ensure a water-tightinterface between membrane and a frame.

It will be understood by those skilled in the art that for eachembodiment of the present invention a degree of experimentation andfine-tuning will be required to achieve optimum results. For example,each different microbial conversion will have a different reactionstoichiometry in that for a given amount of at least one conversionsubstrate to be converted at a given rate, a minimum biomass ofmicrobial culture will be required, and for said minimum biomass ofmicrobial culture to be supported there is a minimum rate at whichprimary growth substrate must be supplied to that culture. For certainmicrobial conversions the exact reaction rate and stoichiometry will beunknown or variable e.g. when a bioreactor according to the presentinvention is used for in-situ bioremediation of contaminated groundwaterthe temperature at the site of microbial conversion will vary and thereaction rate and stoichiometry will vary accordingly. Therefore on-siteexperimentation may be required to establish factors such as the correctrate of supply of primary growth substrate and optionally othernutrients and optimum dimensions of the membranes etc.

The present invention further provides a process of operating abioreactor according to the present invention for conversion of at leastone conversion substrate, which process comprises introducing a solutionof said at least one conversion substrate into the treatment zone,supplying primary growth substrate from the source of primary growthsubstrate through the second permeable membrane at a rate sufficient topromote conversion of said at least one conversion substrate by themicrobial culture, and removing treated solution from the treatmentzone. It is not envisaged that the process of the present invention issuitable for use in medical treatment e.g. in the treatment of human oranimal body-fluids.

In the process of the present invention the solution of at least oneconversion substrate may be introduced into the treatment zone beforeprimary growth substrate is supplied from the source, or primary growthsubstrate may be supplied from the source before the solution of atleast one conversion substrate is introduced into the treatment zone, orboth actions may be initiated simultaneously.

In the process of the present invention the solution of at least oneconversion substrate may conveniently be water contaminated with atleast one contaminant, and may very conveniently be water contaminatedwith at least one alkoxy compound selected from branched alkyl ethersand branched alkyl alcohols.

Accordingly, a preferred embodiment of the present invention provides aprocess wherein the solution of said at least one conversion substrateis water contaminated with at least one alkoxy compound selected frombranched alkyl ethers and branched alkyl alcohols, which processcomprises introducing water contaminated with said at least one alkoxycompound selected from branched alkyl ethers and branched alkyl alcoholsinto the treatment zone and supplying a hydrocarbon growth substratefrom said source through the second permeable membrane at a ratesufficient to promote conversion of said at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols by themicrobial culture, and removing treated solution from the treatmentzone.

A preferred process of the present invention is a process wherein the atleast one alkoxy compound comprises at least one of methyl tert-butylether (MTBE), tert-amyl methyl ether (TAME), ethyl tert-butyl ether(ETBE), diisopropyl ethyl ether (DIPE) or tert-butyl alcohol (TBA), mostpreferably a process wherein the at least one alkoxy compound comprisesmethyl tert-butyl ether (MTBE).

A further preferred process of the present invention is a processwherein the primary growth substrate is at least one hydrocarbon growthsubstrate of up to 12 carbon atoms, e.g. decane, dodecane; morepreferably at least one hydrocarbon growth substrate of up to 9 carbonatoms, e.g. benzene, toluene, ethylbenzene, xylene; more preferably atleast one alkane of up to 6 carbon atoms e.g. methane, ethane, propane,butane, pentane, hexane, and cyclohexane; preferably an alkane of from 2to 6 carbon atoms, more preferably of from 3 to 6 carbon atoms. Anexample of a hydrocarbon growth substrate which may very conveniently beemployed is cyclohexane.

A particularly preferred process of the present invention is a processfor the in-situ treatment of water contaminated with at least one alkoxycompound selected from branched alkyl ethers and branched alkylalcohols, wherein the bioreactor is located in an aquifer containing thewater contaminated with the at least one alkoxy compound.

The present invention further provides a bioreactor for use inbioremediation of contaminated soils and/or groundwaters, preferablyin-situ bioremediation of contaminated soils and/or groundwaters.

FIG. 9 is a plan view of an area of ground containing a gated barriertreatment system for incorporating the second embodiment of FIGS. 4 and5, and the third embodiment of FIGS. 6, 7 and 8.

Whilst the specific embodiments herein described may be used for themicrobial conversion of at least one conversion substrate in solutionfor a variety of applications, they are particularly adapted for thetreatment of water contaminated with methyl tert-butyl ether (MTBE) in aco-metabolic microbial conversion using a hydrocarbon-utilisingmicrobial culture.

Referring to the first embodiment depicted in FIGS. 1, 2 and 3 thebioreactor comprises a first permeable membrane (1) of silicone rubberannular tubing and a second permeable membrane (2) of silicone rubberannular tubing, the tubing of the second permeable membrane beinglocated inside the tubing of the first permeable membrane (1) in aconfiguration which may or may not be co-axial to the first permeablemembrane, a treatment vessel (3), a treatment zone containing watercontaminated with MTBE (4), a culture holding zone (5) containing amicrobial culture (6) capable of metabolising MTBE, a source ofhydrocarbon growth substrate comprising a reservoir of hydrocarbongrowth substrate (8) a supply tube (11) and a hydrocarbon growthsubstrate supply path (7), and a source of nutrient and/or microbialculture comprising a reservoir of nutrient and/or microbial culture (9)and a nutrient delivery tube (13), the first permeable membrane formingan interface between the treatment zone (4) and the culture holding zone(5) and the second permeable membrane forming an interface between thehydrocarbon growth substrate supply path (7) and the culture holdingzone (5), the first permeable membrane being permeable to MTBE butsubstantially impermeable to water and the microbial culture, the secondpermeable membrane being permeable to the hydrocarbon growth substratebut substantially impermeable to water.

The first and second permeable membranes are in the form of heliceslocated within the treatment vessel (3). The supply path (7), whichcorresponds to an annular chamber of the tubing of the second permeablemembrane (2), is connected at a first end to the reservoir ofhydrocarbon growth substrate (8) by the supply tube (11) and at a secondend to an escape tube (12). The culture holding zone (5) is connected ata first end to the reservoir of nutrient and/or microbial culture (9) bythe nutrient delivery tube (13) and at a second end to an effluent tube(15) which passes out of the treatment vessel (3). The nutrient delivery(13) and effluent tubes (15) are associated with a pumping means (14).

The apparatus of FIGS. 1, 2 and 3 is such that when in use watercontaminated with MTBE is introduced into the treatment vessel (3) andhydrocarbon growth substrate is supplied from the source of hydrocarbongrowth substrate through the second permeable membrane (2) at a ratesufficient to promote conversion of the MTBE by the microbial culture(6), which hydrocarbon growth substrate is supplied by passing air or anoxygen enriched gas through the reservoir of hydrocarbon growthsubstrate (8) and passing gaseous growth substrate so generated throughthe supply tube (11) to the supply path (7) where it contacts the secondpermeable membrane (2) and permeates through the membrane to themicrobial culture (6) in the culture holding zone (5). The microbialculture (6) may grow as a biofilm on the surface of the first and/or thesecond permeable membrane or be partially suspended within a nutrientmedium within the culture holding zone (5). Excess gaseous growthsubstrate passes out of the supply path (7) and out of the apparatusthrough the escape tube (12). When necessary, nutrient and/or microbialculture may be delivered to the culture holding zone (5) from thereservoir of nutrient and/or microbial culture (9) via the nutrientdelivery tube (13) using the pumping apparatus (14). The pumpingapparatus (14) may also be used to circulate nutrient and/or microbialculture through the culture holding zone (5) with waste material beingpumped from the culture holding zone and out of the apparatus throughthe effluent tube (15).

As water contaminated with MTBE is introduced into the treatment zone(4) the MTBE will permeate through the first permeable membrane (1) tothe culture holding zone (5) wherein the MTBE contacts the microbialculture (6) and microbial conversion occurs. As the MTBE in the cultureholding zone (5) is converted further MTBE will permeate into theculture holding zone (5), whilst products of microbial conversion otherthan water will permeate out of the culture holding zone (5) back intothe treatment zone (4). When a sufficient amount of the MTBE in thecontaminated water has been converted by the microbial culture (6), thetreated batch of water is drained through a sample port (16) andreplaced with a new batch of contaminated water.

The dimensions of the bioreactor of the first embodiment may beoptimised to obtain maximum performance for the application in which itis to be used. When employed for batch remediation of water contaminatedwith MTBE, the second permeable membrane (2) may typically have a wallthickness of 0.5 mm and an outside diameter of 3 mm and the firstpermeable membrane (1) a wall thickness of 0.5 mm and an outsidediameter of 8 mm.

Referring to the second embodiment depicted in FIGS. 4 and 5 thebioreactor comprises a vessel formed from first permeable membrane (1′)which vessel is a porous plastics cylindrical vessel having a verticalwall (17′) and a concave floor (18′), a second permeable membrane (2′)in the form of helically wound silicone rubber annular tubing whichsecond permeable membrane is positioned within a chamber defined by thecylindrical vessel of the first permeable membrane (1′), a treatmentzone containing groundwater contaminated with MTBE (4′), a cultureholding zone (5′) containing a microbial culture (6′) capable ofmetabolising MTBE, and a source of hydrocarbon growth substratecomprising an above-ground store of gaseous hydrocarbon growth substrate(8′), a supply tube (11′) and a hydrocarbon growth substrate supply path(7′), the first permeable membrane forming an interface between thetreatment zone (4′) and the culture holding zone (5′) and the secondpermeable membrane forming an interface between the hydrocarbon growthsubstrate supply path (7′) and the culture holding zone (5′), the firstpermeable membrane (1′) being permeable to MTBE but impermeable to themicrobial culture and the second permeable membrane being permeable tohydrocarbon growth substrate but substantially impermeable to water.

The supply path (7′), which corresponds to an annular chamber of thetubing of second permeable membrane (2′), is connected at a first end tothe above-ground store of gaseous growth substrate (8′) by the supplytube (11′) and at a second end to an above-ground collection vessel(19′) by a recovery tube (20′), which collection vessel (19′) mayoptionally (not shown in FIG. 4) be connected to the store of gaseoushydrocarbon growth substrate (8′) so as to allow growth substrate to berecycled.

When deployed in an aquifer (21′), the vertical walls (17′) of thecylindrical vessel of the first permeable membrane (1′) extend fromabove the aquifer (21′) (i.e. above a maximum water table level (23′))substantially to the aquifer base (24′).

The bioreactor of the second embodiment further comprises a supplyconduit (26′) which runs from an above-ground store of nutrient and/ormicrobial culture (27′) to the culture holding zone (5′), an extractionconduit (28′) which runs from the culture holding zone (5′) to a secondabove-ground collection vessel (29′), pumping apparatus (14′) forpumping nutrient and/or microbial culture into the culture holding zone(5′) through the supply conduit (26′) and out of the culture holdingzone (5′) through the extraction conduit (28′), a circular-plate gasdiffuser (30′) located near the base of the culture holding zone (5′)and which is connected to an above-ground source of oxygen-containinggas (31′) (e.g. an air pump) by an oxygen delivery conduit (32′).

The cylindrical vessel of first permeable membrane is housed in areceptacle (34′) having a vertical front wall (35′), a vertical backwall (36′) and vertical side walls (37′), which vertical front (35′) andback walls (36′) contain a plurality of slots (39′) through whichcontaminated groundwater (38′) may flow. The receptacle is furtherprovided with impermeable stoppers (25′) to channel groundwater throughthe first permeable membrane.

Referring to FIG. 9, the bioreactor of the second embodiment isespecially suited for use in a gated barrier system as described in U.S.Pat. No. 5,487,622, which gated barrier system may comprise a watertightwall of metal sheets (40′) which are pile driven into an aquifer, whichwall contains a gate (41′) into which a receptacle (34′) containing abioreactor according to the second embodiment may be placed. Thewatertight walls (40′) are positioned in the path of a plume (42′) ofMTBE relative to the flow of groundwater in the aquifer (21′) so as tofunnel contaminated groundwater into the gate (41′).

The bioreactor of the second embodiment is such that when in use in agated barrier system, groundwater contaminated with MTBE (38′) flowsthrough the aquifer and is funnelled by the watertight walls (40′) intothe gate (41′) wherein contaminated groundwater flows through the slots(39′) in the vertical front wall (35′) of the receptacle (34′) and intothe treatment zone (4′) whilst gaseous hydrocarbon growth substrate issupplied from the source of hydrocarbon growth substrate to the secondpermeable membrane (2′) at a rate sufficient to promote conversion ofthe MTBE in the contaminated water in the treatment zone (4′), whichhydrocarbon growth substrate is supplied from the above-ground store ofgaseous hydrocarbon growth substrate (8′) through the supply tube (11′)to the supply path (7′) where it contacts the second permeable membrane(2′) and permeates through the membrane to the microbial culture (6′) inthe culture holding zone (5′). The gaseous hydrocarbon growth substratemay contain pure hydrocarbon growth substrate or may be a mixture ofhydrocarbon growth substrate and oxygen-containing gas.

The microbial culture (6′) may be established in the culture holdingzone (5′) either before or after the bioreactor of the second embodimentis deployed in the aquifer (21′). If established after the bioreactorhas been deployed, the microbial culture (6′) may be established byintroducing an innoculum of microbial culture through the supply conduit(26′) and growing the sample until a required biomass of microbialculture (6′) is established, or alternatively the required biomass ofmicrobial culture (6′) may be grown separately and introduced in totointo the culture holding zone (5′) through the supply conduit (26′). Themicrobial culture (6′) may grow as a biofilm on the surface of the firstand/or the second permeable membrane or may be partially suspendedwithin a medium within the culture holding zone (5′). Nutrient and/oradditional microbial culture (6′) may be supplied to the culture holdingzone (5′) from the above-ground store of nutrient and/or additionalmicrobial culture (27′) through the supply conduit (26′). Similarlyexcess microbial culture (6′) and waste medium may be removed from theculture holding zone (5′) through the extraction conduit (28′) using thepumping apparatus (14′). The material removed from the culture holdingzone (5′) may be analysed to monitor the condition of the microbialculture (6′) in the culture holding zone (5′). Agitation and additionalaeration of the culture holding zone (5′) is achieved by deliveringadditional oxygen-containing gas from the above-ground source ofoxygen-containing gas (31′), through the oxygen delivery conduit (32′)and through the circular plate gas diffuser (30′) into the cultureholding zone (5′).

As groundwater contaminated with MTBE flows into the treatment zone (4′)it comes into contact with the first permeable membrane (1′) so allowinggroundwater contaminated with MTBE (38′) to pass through the firstpermeable membrane into the culture holding zone (5′) where the MTBEcomes into contact with, and is metabolised by, the microbial culture(6′). As MTBE is metabolised in the culture holding zone (5′), furthergroundwater will pass into the culture holding zone (5′), whilst watercomprising microbial conversion products will pass out of the cultureholding zone (5′) into the groundwater in the treatment zone (4′), whichremediated groundwater will flow out of the treatment zone (4′) throughthe slots in the back wall (39′) of the receptacle and into the aquifer(21′) downstream of the bioreactor.

The dimensions of the bioreactor of the second embodiment may beoptimised to attain maximum performance for the application and aquiferin which it is to be used. When employed in a gated barrier system asdescribed in U.S. Pat. No.5,487,622 having a gate width of 1.5 m in anaquifer having a depth of 9 m, the bioreactor may typically comprise asecond permeable membrane (2′) of silicone rubber annular tubing havingan outside diameter of 4 mm and a wall thickness of 0.5 mm, and a firstpermeable membrane (1′) of 5 mm thick porous plastics shaped to form acylindrical vessel having an outside diameter of 0.8 m and verticalwalls (17′) 9.5 m high. The amount of silicone rubber annular tubingpositioned in the cylindrical vessel may be chosen to provide a largeenough surface area of second permeable membrane (2′) for hydrocarbongrowth substrate to permeate through the second permeable membrane (2′)at a rate sufficient to promote conversion of an amount of MTBE in thecontaminated groundwater to be removed by the microbial culture (6′) oralternatively an excess of tubing may be used and a controlled quantityof hydrocarbon growth substrate delivered from the source to promoteconversion of the amount of MTBE to be removed.

Referring to the third embodiment depicted in FIGS. 6, 7 and 8, thebioreactor comprises a plurality of first permeable membranes (1″) inthe form of sheets of silicone rubber, a plurality of second permeablemembranes in the form of sheets of silicone rubber (2″), a plurality ofrectangular frames (51″), a plurality of spacer means (52″) and twovertical side walls (53″ and 54″, which sheets of first and secondpermeable membrane and rectangular frames, and spacer means arevertically positioned parallel with respect to one another in betweenthe two vertical side-walls (53″ and 54″); a plurality of treatmentzones (4″) each defined by boundaries of a channel formed between twoadjacent sheets of first permeable membrane (1″) and a spacer means(52″) or a sidewall, a sheet of first permeable membrane and a spacermeans; a plurality of culture holding zones (5″) containing a microbialculture (6″) capable of metabolising MTBE, each defined by boundaries ofa chamber formed by a sheet of first permeable membrane a sheet ofsecond permeable membrane and a rectangular frame (51″) and a pluralityof primary growth substrate supply paths (7″) each defined by boundariesof a chamber formed between two adjacent sheets of second permeablemembrane, (2″) and a rectangular frame (51″); wherein each sheet offirst permeable membrane (1″) forms an interface between a treatmentzone (4″) and a culture holding zone (5″) and each sheet of secondpermeable membrane (2″) forms an interface between a culture holdingzone (5″) and a primary growth substrate supply path (7″), the firstpermeable membrane being permeable to MTBE but substantially impermeableto water and microbial culture, the second permeable membrane beingpermeable to hydrocarbon growth substrate but substantially impermeableto water.

Deployed in an aquifer, for example in a gated barrier system asdepicted in FIG. 9, the third embodiment is positioned relative to thedirection of groundwater flow such that the channels of the plurality oftreatment zones (4″) are end-on to the flow of groundwater andgroundwater is free to flow into each treatment zone (4″), through thebioreactor, and exit each treatment zone (4″) downstream of the reactor,each treatment zone (4″) being fitted with a spacer means (52″) tomaintain a gap between the sheets of first permeable membrane, each suchspacer means (52″) comprising at least one aperture (64″) through whichgroundwater may enter the treatment zone, a flow path (65″) throughwhich groundwater flows through the treatment zone, and at least oneexit aperture (66″) through which groundwater may exit the treatmentzone downstream of the bioreactor.

The sheets of first and second permeable membrane (1″ and 2″)rectangular frames (51″), two vertical side-walls (53″ and 54″) andspacer means (52″) supporting the first and second permeable membranesare fitted with connection means and conduits such that when assembledthe bioreactor comprises a manifold means for delivery of hydrocarbongrowth substrate to the plurality of hydrocarbon growth substrate supplypaths (7″), said manifold means comprising a hydrocarbon growthsubstrate supply conduit (56″) running from an above-ground store ofgaseous hydrocarbon growth substrate into the top of a first verticalside-wall (53″), a network of hydrocarbon growth substrate distributionconduits (57″) running from the supply conduit (56″) through a top-pieceof each sheet of first and second permeable membrane (1″ and 2″), ofeach rectangular frame (51″) and each spacer means (52″) to eachhydrocarbon growth substrate supply path (7″) and a network of exhaustconduits (59″) which run from each hydrocarbon growth substrate supplypath through the bottom-piece of each sheet of first and secondpermeable membrane (1″ and 2″) each rectangular frame (51″) and eachspacer means (52″) to an escape conduit (60″) which runs out of thesecond vertical side-wall (54″) to an above-ground collection vessel.

The manifold means further comprises a nutrient and/or microbial culturesupply conduit (58″) which runs from an above-ground source of nutrientand/or microbial culture into the top of a first vertical side-wall(53″), a network of nutrient and/or microbial culture distributionconduits (61″) which run through the top-piece of each sheet of firstand second permeable membrane (1″ and 2″), each rectangular frame(51″)and each spacer means (52″) to each culture holding zone (5″), and anetwork of nutrient and/or microbial culture effluent conduits (62″)which run from each culture holding zone (5″) through the bottom-pieceof each sheet of first and second permeable membrane (1″ and 2″), eachrectangular frame (51″) and each spacer means (52″) to a nutrient and/ormicrobial culture recovery conduit (63″) which runs out of the bottom ofthe second vertical side-wall (54″) to a second above-ground collectionvessel. The manifold means may be associated with a pumping means toallow either hydrocarbon growth substrate or nutrient and/or microbialculture to be circulated through the bioreactor.

The bioreactor of the third embodiment is suited for use in a gatedbarrier system as described in U.S. Pat. No. 5,487,622 and mayconveniently be housed in an receptacle as depicted in FIG. 9. Thebioreactor of the third embodiment is such that when in use in a gatedbarrier system water contaminated with MTBE flows through the aquiferand is funnelled by water tight walls into a gate wherein contaminatedwater flows into the receptacle and into the treatment zones (4″) whilstgaseous hydrocarbon growth substrate is supplied from the source ofgaseous hydrocarbon growth substrate to the second permeable membrane(2″) at a rate sufficient to promote the conversion of the MTBE. Gaseoushydrocarbon growth substrate is supplied from an above-ground store ofgaseous hydrocarbon growth substrate via the supply conduit (56″) andthe network of distribution conduits (57″) to the plurality ofhydrocarbon growth substrate supply paths (7″) where it contacts thesecond permeable membranes (2″) and permeates through to the microbialculture (6″) in the plurality of culture holding zones (5″). The gaseoushydrocarbon growth substrate may contain pure hydrocarbon growthsubstrate or may be a mixture of hydrocarbon growth substrate andoxygen-containing gas.

The microbial culture (6″) may grow as a biofilm on the surface of thesheets of first (1″) and/or second permeable membrane (2″) or may bepartially suspended within a medium within the culture holding zone(5″), to which nutrient and/or additional microbial culture may besupplied from an above-ground source of nutrient and/or microbialculture via the supply conduit (58″) and the network of nutrient and/ormicrobial culture distribution conduits (61″). Similarly, excessmicrobial culture (6″) and waste medium may be removed from the cultureholding zone through the network of effluent conduits (62″) and therecovery conduit (63″) to the second above-ground collection vessel byway of a pumping means.

As groundwater contaminated with MTBE flows into the plurality oftreatment zones (4″) it comes into contact with the sheets of firstpermeable membrane (1″) so allowing MTBE in the groundwater to permeatethrough the sheets into a culture holding zone (5″) where it comes intocontact with, and is metabolised by, the microbial culture (6″). As MTBEis metabolised in the plurality of culture holding zones (5″) furtherMTBE will permeate from the groundwater into the plurality of cultureholding zones (5″), whilst products of microbial conversion willpermeate out of the culture holding zones into the treatment zones fromwhere it will flow out of the bioreactor into the aquifer downstream ofthe reactor.

The dimensions of the bioreactor of the third embodiment may beoptimised to attain maximum performance for the application and aquiferin which it is to be used. For convenience, the side-walls andrectangular frames of the third embodiment may be constructed such thattwo or more bioreactors may be assembled on top of one another and/oralongside one another to form a bank or wall of reactors, wherein themanifold means of each bioreactor is further provided with connectorssuch that the manifold means of each bioreactor may be linked so as toform an integrated manifold means through which hydrocarbon growthsubstrate and nutrient and/or microbial culture may be deliveredthroughout each bioreactor simultaneously.

It will be understood by those skilled in the art that the greater thearea of contact between the first permeable membrane and thecontaminated groundwater flowing through the plurality of treatmentzones the greater the efficiency of the bioreactor at removing MTBE fromthe groundwater. It is therefore advantageous that the first and secondpermeable membranes are arranged so as to provide the largest possiblesurface area of contact between first permeable membrane andcontaminated groundwater in the treatment zone whilst still allowing foradequate flow of the groundwater through the bioreactor and adequatesupply of primary growth substrate to, and accommodation of, themicrobial culture.

Typically the bioreactor of the third embodiment may comprise sheets offirst and second permeable membrane of approximately 1 m² , andthickness approximately 0.1 mm, and have a hydrocarbon growth substratesupply path width of 1 mm, a culture holding zone width of from 2-5 mm,and a treatment zone width of from 2-5 mm, the width of a hydrocarbongrowth substrate supply path and a culture holding zone corresponding tothe width of a rectangular frame partially defining its boundaries andthe width of a treatment zone corresponding to the width of a spacermeans partially defining its boundaries.

1. A bioreactor for microbial conversion of at least one conversionsubstrate, which comprises a treatment zone to accommodate when in use asolution of said at least one conversion substrate, a culture holdingzone to accommodate when in use a microbial culture capable ofmetabolising said at least one conversion substrate, a source of primarygrowth substrate for the microbial culture, a first permeable membraneforming an interface between the treatment zone and the culture holdingzone, and a second permeable membrane forming an interface between thesource of primary growth substrate and the culture holding zone, thefirst permeable membrane being of a material which will allow passage ofthe at least one conversion substrate from the treatment zone to theculture holding zone whilst being impermeable to the microbial culture,the second permeable membrane being of a material permeable to theprimary growth substrate but substantially impermeable to water.
 2. Abioreactor according to claim 1 wherein the culture holding zone isdefined by boundaries of a chamber, the treatment zone is external ofthe chamber and the second permeable membrane forms at least a part of ahollow member situated within the chamber.
 3. The bioreactor of claim 1,wherein the conversion substrate is at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols and theprimary growth substrate is at least one hydrocarbon of up to 12 carbonatoms.
 4. The bioreactor of claim 1, wherein the conversion substratecomprises methyl tert-butyl ether and the primary growth substrate is atleast one alkane of up to 6 carbon atoms.
 5. The bioreactor of claim 1,wherein the first permeable membrane is substantially impermeable towater.
 6. A process of operating a bioreactor suitable for conversion ofat least one conversion substrate, which bioreactor comprises atreatment zone to accommodate when in use a solution of said at leastone conversion substrate, a culture holding zone to accommodate when inuse a microbial culture capable of metabolizing said at least oneconversion substrate, a source of primary growth substrate for themicrobial culture, a first permeable membrane forming an interfacebetween the treatment zone and the culture holding zone, and a secondpermeable membrane forming an interface between the source of primarygrowth substrate and the culture holding zone, the first permeablemembrane being of a material which will allow passage of the at leastone conversion substrate from the treatment zone to the culture holdingzone whilst being impermeable to the microbial culture, the secondpermeable membrane being of a material permeable to the primary growthsubstrate but substantially impermeable to water, wherein said processcomprises introducing a solution of said at least one conversionsubstrate into the treatment zone, supplying primary growth substratefrom said source through the second permeable membrane at a ratesufficient to promote conversion of said at least one conversionsubstrate by the microbial culture, and removing treated solution fromthe treatment zone.
 7. A process of claim 6, wherein the solution ofsaid at least one conversion substrate is water contaminated with atleast one alkoxy compound selected from branched alkyl ethers andbranched alkyl alcohols, wherein the primary growth substrate is atleast one hydrocarbon of up to 12 carbon atoms.
 8. A process of claim 7,wherein the at least one alkoxy compound comprises methyl tert-butylether and the hydrocarbon growth substrate is at least one alkane of upto 6 carbon atoms.
 9. A process of claim 7, wherein for the in-situtreatment of water contaminated with at least one alkoxy compoundselected from branched alkyl ethers and branched alkyl alcohols whereinthe bioreactor is located in an aquifer containing the watercontaminated with the at least one alkoxy compound.